In general, the present invention relates to ligation methods, in particular, for joining peptide acceptors to nucleic acids. Methods currently exist for the preparation of RNA-protein fusions. An RNA-protein fusion is created by attaching a peptide acceptor to the 3xe2x80x2 end of an RNA molecule, followed by in vitro or in situ translation of the RNA. The product is a peptide attached to the 3xe2x80x2 end of the RNA encoding it. The generation of these RNA-protein fusions facilitates the isolation of proteins with desired properties from large pools of partially or completely random amino acid sequences, and solves the problem of recovering and amplifying protein sequence information by covalently attaching the RNA coding sequence to its corresponding protein molecule.
The present invention features methods for the attachment of a peptide acceptor to an RNA molecule as well as the RNA-peptide acceptor products. These methods facilitate the production of RNA-protein fusions which can be used, for example, for the isolation of proteins or nucleic acids with desired properties from large pools of partially or completely random amino acid or nucleic acid sequences. This inventive method may be carried out by a variety of strategies for affixing a peptide acceptor to a nucleic acid molecule. These various approaches differ from one another in the types of bonds formed by the attachment of the peptide to the nucleic acid, and in the reagents used to achieve the attachment.
Accordingly, in a first aspect, the invention features a method for affixing a peptide acceptor to an RNA molecule involving providing an RNA molecule having a 3xe2x80x2 sequence which forms a hairpin structure, providing a peptide acceptor covalently bonded to a nucleic acid linker molecule, and hybridizing the RNA molecule to the nucleic acid linker molecule under conditions which allow covalent bond formation to occur between the peptide acceptor and the RNA molecule.
In a second aspect, the invention features a method for affixing a peptide acceptor to an RNA molecule involving providing a peptide acceptor having a linker with a 5xe2x80x2 sequence that forms a hairpin, hybridizing the peptide acceptor to the RNA molecule, and covalently bonding the peptide acceptor to the RNA. In one embodiment of the above aspects of the invention, the peptide acceptor is bonded to the RNA molecule using T4 DNA ligase.
In a third aspect, the invention features a method for attaching a peptide acceptor to an RNA molecule, by providing an RNA molecule and a peptide acceptor covalently bonded to a linker molecule, where the linker molecule initiates with a deoxynucleotide triphosphate or dideoxynucleotide triphosphate, and contacting the RNA molecule and peptide acceptor with terminal deoxynucleotidyl transferase to covalently bond the peptide acceptor to the RNA molecule.
In a fourth aspect, the invention features a method of affixing a peptide acceptor to an RNA molecule by chemically ligating the RNA molecule to the peptide acceptor.
In one embodiment of this aspect, the peptide acceptor is joined to a psoralen moiety and crosslinked to the RNA molecule via the psoralen moiety. The psoralen moiety may be attached to either the 5xe2x80x2 or 3xe2x80x2 end of a linker molecule which is itself attached to the peptide acceptor, or the psoralen moiety may be located at an internal position of the linker molecule. According to this technique, the peptide acceptor is crosslinked to the RNA molecule using UV irradiation. In further embodiments of this particular aspect, the psoralen is attached to the peptide acceptor through a C6 alkyl chain and/or the RNA molecule contains a stop codon positioned proximal to its 3xe2x80x2 end. Preferably, the linker is between 25 and 40 nucleotide units in length. In addition, prior to crosslinking the peptide acceptor to the RNA molecule, the RNA may be hybridized to a linker that further includes a photocleavable moiety. The hybridized RNA may then be immobilized to a solid support through the photocleavable moiety. Preferably, the photocleavable moiety is biotin.
In another embodiment of the fourth aspect of the invention, the RNA molecule is functionalized and is attached to a peptide that has been suitably modified to permit chemical bond formation between the peptide acceptor and the RNA molecule. Preferably, the RNA molecule is functionalized through IO4xe2x88x92oxidation. The peptide acceptor may be functionalized by attaching a molecule to the peptide acceptor chosen from the group consisting of amines, hydrazines, (thio)hydrazides, and (thio)semicarbazones.
In yet another embodiment of the fourth aspect of the invention, the chemical ligation is carried out in the absence of an external template. Alternatively, the chemical ligation reaction can be carried out in the presence of an external template. This second method involves aligning the RNA molecule and the linker portion of a peptide acceptor using a template, so that the 5xe2x80x2 end of the template hybridizes to the linker portion of the peptide acceptor and the 3xe2x80x2 end of the template hybridizes to the RNA molecule. The chemical ligation of an RNA molecule to a peptide acceptor can also be carried out in the absence of an external template by hybridizing the linker molecule itself, which is covalently bonded to the peptide acceptor, to the RNA molecule. This hybridization brings the peptide acceptor and RNA molecule into close proximity for ligation. Preferably, the functional group is at the 5xe2x80x2 end of the linker region of the peptide acceptor, or is flanked by a hybridization domain on one side and the peptide acceptor on the other side.
In a further embodiment of the fourth aspect of the invention, the chemical ligation of the peptide acceptor to the RNA molecule involves attaching a functional group to the RNA molecule through reductive amination of the RNA, followed by modification of the peptide acceptor to react with the RNA molecule. The two molecules are then joined through formation of a covalent bond. Preferably, the functional group attached to the RNA molecule is a thiol, maleimide, or amine.
In a fifth aspect, the invention features a method for attaching a peptide acceptor to an RNA molecule through a non-covalent bond. In one embodiment, the attachment is achieved by covalently bonding a peptide nucleic acid (PNA) to the peptide acceptor and non-covalently bonding the peptide acceptor to the RNA molecule through the PNA. In this embodiment, the RNA molecule may contain a stop codon.
In yet other aspects, the invention features RNA molecules chemically or non-covalently ligated to peptide acceptors as well as the nucleic acid-protein fusions generated by transcription and translation (and, if desired, reverse transcription and/or amplification) of these RNA molecules. In one embodiment, the peptide acceptor is ligated at the 3xe2x80x2 end of the RNA molecule.
In still another aspect, the invention features methods for the selection of a desired protein or nucleic acid using the RNA-peptide acceptor molecules of the invention. The selection techniques utilize the present molecules for RNA-protein fusion formation, and subsequent selection of proteins or nucleic acids of interest. The selection methods may be carried out by any of the approaches described, for example, in Szostak et al., WO 98/31700, and Szostak et al., U.S. Ser. No. 09/247,190 now U.S. Pat. No. 6,261,804, hereby incorporated by reference.
In a final aspect, the invention features a method of generating an RNA-protein fusion. This method involves providing an RNA molecule hybridized to a linker, where the linker contains a photocleavable moiety, a psoralen moiety, and a peptide acceptor; immobilizing the RNA to a solid support under conditions in which non-immobilized RNA are substantially removed from the support; crosslinking the peptide acceptor to the RNA, through the psoralen moiety, whereby this crosslinking simultaneously releases the crosslinked RNA from the solid support; and translating the crosslinked RNA to form an RNA-fusion protein. In one embodiment, the photocleavable moiety is biotin.
In all of the above aspects of the invention, the RNA molecule may include a translation initiation sequence and a start codon operably linked to a candidate protein coding sequence. In addition, one preferred peptide acceptor is puromycin, a nucleoside analog which adds to the C-terminus of a growing peptide chain and terminates translation. In one embodiment, the peptide acceptor includes puromycin attached to a linker, for example, a nucleotide linker. This linker facilitates the alignment of the peptide acceptor to the RNA molecule for attachment. In a further embodiment, the linker region of the peptide acceptor includes non-nucleotide moieties, for example, PEG. Other possible choices for acceptors include tRNA-like structures at the 3xe2x80x2 end of the RNA, as well as other compounds that act in a manner similar to puromycin. Such compounds include, without limitation, any compound which possesses an amino acid linked to an adenine or an adenine-like compound, such as the amino acid nucleotides, phenylalanyl-adenosine (A-Phe), tyrosyl adenosine (A-Tyr), and alanyl adenosine (A-Ala), as well as amide-linked structures, such as phenylalanyl 3xe2x80x2 deoxy 3xe2x80x2 amino adenosine, alanyl 3xe2x80x2 deoxy 3xe2x80x2 amino adenosine, and tyrosyl 3xe2x80x2 deoxy 3xe2x80x2 amino adenosine; in any of these compounds, any of the naturally-occurring L-amino acids or their analogs may be utilized. In addition, a combined tRNA-like 3xe2x80x2 structure-puromycin conjugate may also be used in the invention.
In one preferred design of the invention, a DNA sequence is included between the end of the message and the peptide acceptor. This sequence is designed to cause the ribosome to pause at the end of the open reading frame, providing additional time for the peptide acceptor (for example, puromycin) to accept the nascent peptide chain before hydrolysis of the peptidyl-tRNA linkage. During in vitro translation the ribosome may also pause at the site of chemical ligation, especially at a psoralen crosslinking site or at a PNA clamp.
In another preferred design of the invention, predominantly non-nucleotide linker moieties may be used in place of the nucleotide linkers attached to the peptide acceptor. This design facilitates the ligation of a peptide acceptor to an RNA molecule. For example, the linker may contain triethylene glycol spacers. The linker may also contain 2xe2x80x2-OMe-RNA phosphoramidites. In some cases where hybridization is a prerequisite for chemical or enzymatic ligation, a sufficient portion of the linker next to the ligation site must be comprised of nucleic acids. Furthermore, the RNA or linker of the invention may contain a sequence (e.g., a poly(A) sequence) for use in purification, for example, affinity purification of the RNA or an RNA-protein fusion molecule formed from such an RNA or linker.
In addition, in all of the above aspects of the invention the RNA molecule affixed to a peptide acceptor may be in vitro or in situ translated to produce an RNA-protein fusion molecule. The RNA-protein fusion molecule is then incubated in the presence of high salt and/or incubated at low temperature (e.g., overnight at xe2x88x9220xc2x0 C.) as described by Szostak et al. (09/247,190, now U.S. Pat. No. 6,261,804). The RNA-protein fusion molecule may be also purified, for example, using standard poly(A) purification techniques.
As used herein, by a xe2x80x9cproteinxe2x80x9d is meant any two or more naturally occurring or modified amino acids joined by one or more peptide bonds. xe2x80x9cProteinxe2x80x9d and xe2x80x9cpeptidexe2x80x9d are used interchangeably.
By an xe2x80x9cRNAxe2x80x9d is meant a sequence of two or more covalently bonded, naturally occurring or modified ribonucleotides. One example of a modified RNA included within this term is phosphorothioate RNA.
By a xe2x80x9ctranslation initiation sequencexe2x80x9d is meant any sequence that is capable of providing a functional ribosome entry site. In bacterial systems, this region is sometimes referred to as a Shine-Dalgamo sequence.
By a xe2x80x9cstart codonxe2x80x9d is meant three bases which signal the beginning of a protein coding sequence. By a xe2x80x9cstop codonxe2x80x9d is meant three bases which signal the termination of a protein coding sequence. Generally, start codons are AUG (or ATG) and stop codons are UAA (or TAA), UAG (or TAG), or UGA (or TGA); however, any other base triplets capable of being utilized as start or stop codons may be substituted.
By xe2x80x9ccovalently bondedxe2x80x9d is meant joined together either directly through a covalent bond or indirectly through another covalently bonded sequence (for example, DNA corresponding to a pause site).
A By xe2x80x9cnon-covalently bondedxe2x80x9d is meant joined together by means other than a covalent bond.
By a xe2x80x9chairpin structurexe2x80x9d is meant a double-stranded region formed by a single nucleic acid strand. Preferably, such hairpin structures are at least 8 base pairs in length, and more preferably, between 8 and 15 base pairs in length.
By xe2x80x9cchemically ligatingxe2x80x9d is meant the joining together of two molecules without the use of an enzyme. Chemical ligation can result in non-covalent as well as covalent bonds.
By a xe2x80x9cpeptide acceptorxe2x80x9d is meant any molecule capable of being added to the C-terminus of a growing protein chain by the catalytic activity of the ribosomal peptidyl transferase function. Typically, such molecules contain (i) a nucleotide or nucleotide-like moiety, for example adenosine or an adenosine analog (di-methylation at the N-6 amino position is acceptable), (ii) an amino acid or amino acid-like moiety, such as any of the 20 D- or L-amino acids or any amino acid analog thereof including 0-methyl tyrosine or any of the analogs described by Ellman et al. (Meth. Enzymol. 202:301, 1991), and (iii) a linkage between the two (for example, an ester, amide, or ketone linkage at the 3xe2x80x2 position or, less preferably, the 2xe2x80x2 position). Preferably, this linkage does not significantly perturb the pucker of the ring from the natural ribonucleotide conformation. Peptide acceptors may also possess a nucleophile, which may be, without limitation, an amino group, a hydroxyl group, or a sulfhydryl group. In addition, peptide acceptors may be composed of nucleotide mimetics, amino acid mimetics, or mimetics of the combined nucleotide-amino acid structure.
By a xe2x80x9clinkerxe2x80x9d or xe2x80x9clinker moleculexe2x80x9d is meant a sequence that includes deoxyribonucleotides, ribonucleotides, or analogs thereof By xe2x80x9cfunctionalizexe2x80x9d is meant to modify in a manner that results in the attachment of a functional group or moiety. For example, an RNA molecule may be functionalized through IO4xe2x88x92oxidation or amination, or a peptide acceptor may be functionalized by attaching an amine, hydrazine, (thio)hydrazide, or (thio)semicarbazone group.
By an xe2x80x9cexternal template,xe2x80x9d is meant a nucleic acid sequence which is added to a ligation reaction mixture, but which is not a part of the final product of the ligation reaction.
By xe2x80x9chigh saltxe2x80x9d is meant having a concentration of a monovalent cation of at least 200 mM, and, preferably, at least 500 mM or even 1 M, and/or a concentration of a divalent or higher valence cation of at least 25 mM, preferably, at least 50 mM, and, most preferably, at least 100 mM.
By xe2x80x9caffinity purification sequencexe2x80x9d is meant a nucleotide sequence that is utilized in the purification of a nucleic acid or a nucleic acid-protein fusion molecule. For example, an affinity purification sequence may be a poly(A) sequence, such as A8-20 (SEQ ID NOS: 16-26), which can be used for purification of nucleic acid or fusion molecules on oligo-dT cellulose. An affinity purification sequence may also be a polypeptide sequence that is used to purify a nucleic acid-protein fusion molecule. Other exemplary purification techniques are described by Szostak et al. U.S. Ser. No. 09/247,190, now U.S. Pat. No. 6,261,804.
The present invention provides a number of advantages. For example, the methods described herein facilitate the efficient ligation of peptide acceptors to RNA molecules, in some aspects, without the need for an external template to bring the RNA and peptide acceptor together. The invention also reduces the cost associated with the generation of an RNA-protein fusion.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.
The drawings will first briefly be described.